Methods of treating a latent hiv-1 infection using non-coding deoxyribonucleic acids

ABSTRACT

The invention relates to a non-coding sequence of deoxyribonucleic acids comprising at least one sequence motif N 1 N 2 CGN 3 N 4 , wherein N is a nucleotide comprising A, C, T, or G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine for the treatment of viral infections. In particular, the non-coding sequence of deoxyribonucleic acids is used in combination with antiretroviral therapy and/or histone de-acetylase inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/760,424, which is a National Stage of International Application No. PCT/EP2016/072385, filed Sep. 21, 2016, claiming priority based on Great British Patent Application No. 1516676.2, filed Sep. 21, 2015.

SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporated by reference in its entirety. The accompanying file, named 052838-574C01US.xml was created on Jan. 4, 2023 and is 8 KB bytes.

DESCRIPTION Field of the Invention

The invention relates to a combination and its use for the treatment of diseases like viral infections.

Brief Description of the Related Art

The term “immunotherapy” defines the treatment of diseases by stimulating, inducing, enhancing or suppressing an immune response. The strategy of immunotherapies is to fight diseases, such as cancer, infectious diseases, allergy and asthma by activating the immune system.

Viral infection may result in a state called viral latency, which is characterized by a state of reversible non-productive infection of individual cells. Viral latency provides an important mechanism for viral persistence and escape from immune recognition (Perng and Jones, Interdiscip Perspect Infect Dis, 2010, p 262415).

It is known that Herpes viruses for instance use genetic programs that allow persistence of their viral genomes with minimal viral gene expression. For retroviruses, stable integration of reverse transcribed viral cDNA into the host cell genome is an important step towards the persistence of viral genomes for the lifespan of the infected cells. There are retroviruses which establish a state of latent infection. The term latency was initially used for HIV-1 in the clinical sense to describe the long asymptomatic period between initial infection and development of AIDS. However, it became clear that HIV-1 replicates actively throughout the progress of infection, even during the asymptomatic period. One of the major mechanism by which HIV-1 escapes from immune response is through rapid evolution of escape mutations that abrogate recognition by neutralizing antibodies and cytolytic T lymphocytes (Bailey et al., Curr Opin Immunol 16, p. 470-476, 2004). Nevertheless, it has become clear that HIV-1 can establish a state of latent infection at the level of individual T cells. Thus, the removal of the latency reservoir could be a major factor in improving the therapy of HIV.

Antiretroviral therapy (ART) refers to medications for the treatment of retroviral infections like HIV infections. The drugs do not abolish the virus so that patients will be virus-free after treatment. However, when the drugs—or a selection of them is given in combination they may prevent the growth of the virus. When the virus is slowed down, so is HIV disease. Antiretroviral drugs are referred to as ARV. Combination ARV therapy (cART) is referred to as highly active ART (HAART).

ARV drugs attack HIV in different ways and they are divided in so-called classes. The first class of anti-HIV drugs which were developed are the “nucleoside reverse transcriptase inhibitors” (also called NRTIs or “nukes”.) These drugs block the use of HIV's genetic material to create DNA from RNA. AZT is the most prominent member of this class.

Another class are the “non-nucleoside reverse transcriptase inhibitors”, also called non-nukes or NNRTIs, which also block the transcription of RNA into DNA.

Members belonging to the class of “protease inhibitors” block the step of cutting raw material for new HIV virus into specific pieces. Finally, there is the class of “entry inhibitors” preventing HIV from entering a cell, and the class of “HIV integrating inhibitors”, preventing the integration of HIV genetic material into the host genome. Within the meaning of the present disclosure cART refers to the combinatorial use of at least two member of these classes of ARV.

Recently it was shown that the state of HIV-1 latency can be disrupted safely in patients with combination antiretroviral therapy (cART) by latency reversing agents (LRA), particularly histone de-acetylase inhibitors (HDACi) (Archin et al., 2012, Nature 487(7408):482-485; Rasmussen et al., 2013, Hum Vaccin Immunother 9(5)). However, it became obvious in the majority of patients that reactivation of latently infected cells alone does not reduce the HIV-1 reservoir. This seems to be related to insufficient immune mediated killing of reactivated cells. However, the activation of the innate and adaptive immunity bears the risk of activating HIV-infected cells so that the disease will progress. Thus, it is questionable whether the activation of the immune system will help HIV patients.

The concept of immunotherapy-supported HIV treatment is supported by a recent in vitro study showing that CD8+ T cells from cART-treated aviremic HIV patients are capable of killing reactivated infected autologous CD4+ T cells but only when the CD8+ T cells have been antigen-stimulated prior to co-culture (Shan et al., 2012, Immunity 36(3):491-501).

Thus, there is a need for compounds which will expose the latent reservoir of HIV infected cells through their activation and targeting of the reactivated cells for immune mediating killing through pharmacological interventions.

Based on this state of the art, the objective of the instant disclosure is to provide immunostimulating compounds for reactivating latent HIV infected cells and their recognition and destruction by the immune system, and their use as a medicament.

SUMMARY OF THE INVENTION

With regard to the prior art it is an objective of the instant disclosure to provide a non-coding sequence of deoxyribonucleic acids comprising at least one sequence motif N¹N²CGN³N⁴, wherein N is a nucleotide comprising A, C, T, or G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine for the treatment of viral infections.

It is intended to use the non-coding sequence of deoxyribonucleic acids of the present invention in combination with combination antiretroviral therapy and/or histone de-acetylase inhibitors in viral infections, wherein the viral infection may be latent.

The non-coding sequence of deoxyribonucleic acids of the instant disclosure is intended to be provided for the treatment of HIV-1, in particular for the treatment of latent HIV-1 infection.

With the non-coding sequence of deoxyribonucleic acids of the present disclosure, N¹N² may be an element taken from the group of GT, GG, GA, AT and AA, N³N⁴ may be an element taken from the group of CT, TG and TT.

The non-coding sequence of deoxyribonucleic acids can be either linear open-chained on both sides, linear open-chained on one side of a double stranded part with a single stranded hairpin on the respective other side of the double strand or a dumbbell-shaped partially single-stranded covalently closed chain of deoxyribonucleic acids.

The non-coding sequence of deoxyribonucleic acids may comprise at least three of said sequence motifs N¹N²CGN³N⁴.

The non-coding sequence of deoxyribonucleic may further be a linear open-chained non-coding sequence of deoxyribonucleic acids comprising at least one nucleotide in L-conformation.

At least one of the five terminal nucleotides located at the 5′- and/or the 3′-end of a DNA single strand of the linear open-chained non-coding sequence of deoxyribonucleic acids is in L-conformation. It is obvious for a person having ordinary skill in the art that this restriction to the terminal ends means that only within the terminal five nucleotides either on the 5′- and/or the 3′-end nucleotides in 1-confirmation are contained.

It is intended that the non-coding sequence of deoxyribonucleic acids may comprise at least one of the sequence of

(SEQ ID NO: 1) a. GTTCCTGGAG ACGTTCTTAG GAACGTTCTC CTTGACGTTG GAGAGAAC or (SEQ ID NO: 2) b. ACCTTCCTTG TACTAACGTT GCCTCAAGGA AGGTTGATCT TCATAACGTT GCCTAGATCA, or (SEQ ID NO: 3) c. AACGTTCTTCGGGG CGTT, or (SEQ ID NO: 4) d. AGGTGGTAAC CCCTAGGGGT TACCACCTTC ATCGTCGTTT TGTCGTTTTG TCGTTCTT.

The non-coding sequence of deoxyribonucleic acids of the instant disclosure may have a length of 40 to 200 nucleotides or more specifically 48 to 116 nucleotides.

The above shown sequence of AACGTTCTTCGGGG CGTT may be part of the sequence CCTAGGGGTT ACCACCTTCA TTGGAAAACG TTCTTCGGGG CGTTCTTAGG TGGTAACC CCTAGGGGTT ACCACCTTCA TTGGAAAACG TTCTTCGGGG CGTTCTTAGG TGGTAACC (SEQ ID NO:5).

It is intended that the sequence motif N¹N²CGN³N⁴ may be part of a single stranded region of the non-coding sequence of deoxyribonucleic acids.

Another object of the instant disclosure is a method comprising the step of providing a non-coding sequence of deoxyribonucleic acids of the present disclosure simultaneously, alternating or successively with combination antiretroviral therapy and/or histone de-acetylase inhibitors.

The non-coding sequence of deoxyribonucleic acids may be provided prior to combination antiretroviral therapy and/or histone de-acetylase inhibitors. Alternatively, it is intended that the non-coding sequence of deoxyribonucleic acids may be provided alternatingly with combination antiretroviral therapy and/or histone de-acetylase inhibitors.

A further object of the instant disclosure is a non-coding sequence of deoxyribonucleic acids with the above disclosed features for use as a medicament.

The use of a non-coding sequence of deoxyribonucleic acids of the instant disclosure for the manufacture of a pharmaceutical composition comprising the non-coding sequence of deoxyribonucleic acids and possibly additionally ARV and/or histone de-acetylase inhibitors is another object of the instant disclosure.

The pharmaceutical composition may be a vaccine and it is intended to use the non-coding sequence of deoxyribonucleic acids with the features as disclosed above as an adjuvant in therapeutic or prophylactic vaccination for the treatment of HIV-1.

BRIEF DESCRIPTION OF THE FIGURES

The disclosure will be further illustrated by examples and figures without being limited to the disclosed embodiments. A non-coding sequence of deoxyribonucleic acids of the present invention is labelled “MGN1703” in FIG. 1-3 , and as “dSLIM” in FIG. 4 . It shows:

FIG. 1A/B MGN1703 upregulates CD69 expression, a marker of cell activation, on peripheral blood immune effector cells (natural killer cells and CD8 T cells)

FIG. 2 MGN1703 stimulated NK cells inhibit the spread of HIV

FIG. 3 Non-coding DNA constructs of the present invention cause negligible changes in pro-inflammatory cytokines IL-6 and TNF α.

FIG. 4A/B Activating effect of stimulation of NK cells with non-coding immunostimulating sequence of DNA

FIG. 5A/B/C Treatment of NK cells with non-coding DNA constructs. The non-coding DNA constructs enhance NK cell-mediated killing of HIV-1 producing autologous CD4+ T cells.

DESCRIPTION OF THE INVENTION

The instant invention provides a compound and method for the treatment of viral infections. In particular, the invention relates to retroviral infection and the treatment of viral latency. The provided non-coding sequences of deoxyribonucleotides shall substantially augment NK cell activity and HIV-specific CD8+ cell cytotoxic activity leading to enhanced killing of HIV-expressing cells.

Within the meaning of the present disclosure a linear open-chained DNA sequence is designated as oligonucleotide, abbreviated with ODN. Said DNA sequence can be single-stranded or partially or completely double-stranded. The terms oligo, oligonucleotide and oligodeoxynucleotide are used synonymously and do not indicate a limitation of the length of the corresponding DNA sequence. The single components of oligonucleotides are nucleotides.

An oligo can be manufactured synthetically or be partially or completely of biological origin, wherein a biological origin includes genetically based methods of manufacture of DNA sequences.

L-DNA or nucleotides in L-conformation refer to nucleotides, which comprises L-deoxyribose as the sugar residue instead of the naturally occurring D-deoxyribose. L-deoxyribose is the enantiomer (mirror-image) of D-deoxyribose. Oligonucleotides partially or completely consisting of nucleotides in L-conformation can be partially or completely single- or double-stranded; however, nucleotides in L-conformation cannot hybridize to nucleotides in D-conformation (Hauser et al., Nucleic Acid Res. 2006 34: 5101-11). L-DNA is equally soluble and selective as D-DNA. Yet, L-DNA is resistant towards enzymatic exoactivity of naturally occurring enzymes, especially exonucleases, so L-DNA is protected against intracellular degradation (Urata et al., Nucleic Acids Res. 1992 20: 3325-32). Therefore, L-DNA is very widely applicable.

A “stem” according to the present disclosure shall be understood as a DNA double strand formed by base pairing either within the same oligonucleotide (which is then partially self-complementary) or within different oligonucleotides (which are partially or completely complementary). Intramolecular base-pairing designates basepairing within the same oligonucleotide and base-pairing between different oligonucleotides is termed as intermolecular base-pairing.

A “loop” within the meaning of the present disclosure shall be understood as an unpaired, single-stranded region either within or at the end of a stem structure. A “hairpin” is a distinct combination of a stem and a loop, which occurs when two self-complementary regions of the same oligonucleotide hybridize to form a stem with an unpaired loop at one end.

A “solid phase” to which the nucleotides are covalently or non-covalently attached refers to, but is not restricted to, a column, a matrix, beads, glass including modified or functionalized glass, silica or silica-based materials including silicon and modified silicon, plastics (comprising polypropylene, polyethylene, polystyrene and copolymers of styrene and other materials, acrylics, polybutylene, polyurethanes etc.), nylon or nitrocellulose, resins, polysaccharides, carbon as well as inorganic glasses and plastics. Thus, microtiter plates are also within the scope of a solid phase according to the present disclosure.

Immunomodulation according to the present disclosure refers to immunostimulation and immunosuppression Immunostimulation means preferentially that effector cells of the immune system are stimulated in order to proliferate, migrate, differentiate or become active in any other form. B cell proliferation for instance can be induced without co-stimulatory signals by immunostimulatory oligonucleotides, which normally require a co-stimulatory signal from helper thymocytes.

Immunosuppression on the other hand shall be understood as reducing the activation or efficacy of the immune system Immunosuppression is generally deliberately induced to prevent for instance the rejection of a transplanted organ, to treat graft-versus-host disease after a bone marrow transplant, or for the treatment of autoimmune diseases such as, for example, rheumatoid arthritis or Crohn's disease.

An agonist within the meaning of the instant disclosure and in accordance with its common definition represents a chemical or molecule that binds to another molecule, like a receptor or ligand and thus activates the molecule. In contrast to an agonist that activates, an antagonist shall be understood as a chemical or molecule that blocks the interaction of the molecule to which the antagonist binds with a respective agonist. Depending on the context, an antagonist in the understanding of the instant invention may also result in the activation of a process, because the antagonist blocks the interaction of another antagonist with a receptor for instance.

The term “pharmaceutically applicable or acceptable salts” as used herein includes salts of a compound of the combination, which are prepared with relatively nontoxic (i.e. pharmaceutically acceptable) acids or bases, depending on the particular substituents found on the compounds of the present invention. If, for example, compounds of the present invention contain acidic functionalities, base addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. If compounds of the present invention contain basic functionalities, acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, phosphoric, partially neutralized phosphoric acids, sulfuric, partially neutralized sulfuric, hydroiodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like. Certain specific compounds of the present invention may contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Contacting the salt with a base may regenerate the neutral forms of the compounds of the present invention or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention. The compounds of the present invention may possess chiral or asymmetric carbon atoms (optical centers) and/or double bonds. The racemates, diastereomers, geometric isomers and individual optical isomers are encompassed by the present invention. The compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are also encompassed by the present invention. The compounds of the present invention may furthermore exist in multiple crystalline or amorphous forms.

Deoxyribonucleic acid molecules, consisting of a partially single-stranded, dumbbell-shaped, covalently closed chain of deoxyribonucleoside residues, which contain one or more sequences of the base sequence N¹N²CGN³N⁴, wherein N¹N² is an element from the GT, GG, GA, AT or AA group, N³N⁴ is an element from the CT or TT group, as well as C deoxycytosine, G deoxyguanosine, A deoxyadenosine and T deoxythymidine, shall be used in combination with antiretroviral drugs and/or histone de-acetylase inhibitors.

The deoxyribonucleic acid molecules relating to the instant disclosure may have a length of up to 200 nucleotides. In particular, sequences with a length between 48 and 116 nucleotide are intended.

The dumbbell-shaped non-coding sequences of deoxyribonucleic acid molecules may comprise the base sequence N¹N²CGN³N⁴ is their single-stranded regions.

The immunostimulation may take place in vitro, ex vivo or in vivo.

The instant disclosure provides also linear open-chained DNA sequence comprising at least one CpG motif and at least one nucleotide in L-conformation. Due to the partial/complete L-conformation, the DNA sequence is partially or fully resistant to exonucleases. In case that the construct has on one end of a double strand a single stranded-loop, the end is also protected against degradation. Thereby, the ODNs are in total protected against cellular degradation without having the need to use a phosphorothioate backbone, which has been shown to be toxic. In addition, the ODNs only consist of a minimum number of nucleotides, which makes them small and thereby easy to transfect into cells.

The non-coding sequence of deoxyribonucleic acids comprising at least one sequence motif N¹N²CGN³N⁴ can be single-stranded or partially or completely double-stranded. This includes base-pairing within the same molecule (intramolecular) or within different molecules (intermolecular) or any combination thereof. It is also possible that the construct comprises at least one unpaired, single-stranded region. As a further embodiment, hairpin structures are included. Due to the partial or complete L-conformation, a longer half-life of the construct is ensured as nucleotides in L-conformation are not subject to degradation.

It is also within the scope of the instant disclosure that at least two molecules, which are single-stranded or partially or completely double-stranded can ligate to each other to form multimeric constructs. These multimeric constructs thus incorporate at least as many CpG motifs as ligation partners, tightly packed within one molecule, and are therefore expected to elicit also a considerable immune response as part of the combination with T-cell regulators. The resulting single-stranded or partially or completely double-stranded multimeric constructs can either be covalently closed comprising nucleotides in L-conformation within the molecule or open multimeric constructs comprising nucleotides in L-conformation at the 5′- and/or the 3′-end for protection against cellular degradation.

The disclosure further comprises chemical modifications of at least one nucleotide in the non-coding sequence of deoxyribonucleic acids comprising at least one sequence motif N¹N²CGN³N⁴ with a functional group selected from the group comprising carboxyl, amine, amide, aldimine, ketal, acetal, ester, ether, disulfide, thiol and aldehyde groups. This allows coupling of the DNA construct to a compound selected from the group comprising peptides, proteins, carbohydrates, antibodies, synthetic molecules, polymers, micro projectiles, metal particles or a solid phase by, for example, adsorption, covalent or ionic bonding.

The modification can be specifically selected for the respective purpose. The construct can thus be used, for example, to shuttle other molecules to the specific cell responding to the CpG motif/s incorporated. In addition, it is possible by such modifications to couple the construct to micro projectiles, which can be used to transfer the construct into the cell. The construct can also be coupled to a solid phase, e. g. a microtiter plate.

FIG. 1 shows that MGN1703 upregulates CD69 expression, a marker of cell activation, on peripheral blood immune effector cells. Total PBMC (n=5 separate donors) were stimulated with MGN1703 or LPS. Following stimulation, cells were analyzed via flow cytometry and CD69 expression on NK cells (left) and CD8+ T cells (right) were characterized. Depicted is mean fold above media control.

FIG. 2 shows that MGN1703 stimulated NK cells inhibit the spread of HIV. CD4-depleted PBMC (one representative donor with SD shown) were stimulated with MGN1703 or LPS. Following stimulation, the cells were incubated with autologous HIV-infected CD4+ cells. HIV capsid protein (Gagp24) was quantitated in culture supernatants over time as a measure of virus spread.

FIG. 3 shows that the non-coding DNA constructs have almost no effect on the pro-inflammatory cytokines IL-6 (FIG. 3A) and TNFα (FIG. 3B). “UT” denotes untreated controls, “MGN1703” denotes the non-coding DNA constructs applied in concentrations of 0.75 μM, 1.5 μM, and 3 μM from left to right. Thus, the use of a non-coding immunostimulating sequence of deoxyribonucleotides activated viral infected cells without initiating an unintended cytokine based inflammation.

In order to confirm the activating effect of stimulating NK cells with non-coding immunostimulating sequence of deoxyribonucleotides, the effect on activating or inhibitory receptors of NK cells was investigated. FIGS. 4A and 4B show that only the expression of activating receptors is increased by the non-coding DNA sequences.

FIG. 5 shows the results of experiments that were conducted to confirm that the viral inhibition shown in FIG. 2 was a result of HIV-specific killing and not unspecific CD4 T cell death. HIV infected cells were collected on day 6 and stained for intracellular P24 antigen. FIG. 5A shows CD4 T-cells which were cultured alone, FIG. 5B shows CD4 T-cells cultured together with untreated NK cells and FIG. 5C shows CD4 T-cells cultured together with dSLIM treated NK cells. The square in each figure gates the population of HIV infected cells. Their percentage on the total population is indicated above the square.

The results in FIG. 5 confirm that the treatment of NK cells with non-coding DNA constructs according ton the present disclosure is suitable to reduce considerably the number of HIV infected cells.

In essence, the instant disclosure demonstrates that the treatment of viral infections leading to latency can be supported by the treatment with non-coding DNA constructs according to the present disclosure. Infected cells will be activated or “demasked” so that the immune system will be able to kill them. Thus the combinatorial treatment of HIV infections with cART for instance is supported by applying immunostimulating non-coding DNA.

Referring to the results described as prior art where cART and HDACi were combined, it seems to be possible that the combinatorial treatment of those two components of HIV treatment with immunostimulating non-coding DNA constructs will be beneficial for the HIV treatment. Corresponding experiments aiming at proving such a beneficial effect are currently performed.

It has to be noted that the non-coding immunostimulating DNA constructs of the instant disclosure have the advantage of avoiding pro-inflammatory cytokine stimulation. In contrast, non-coding DNA constructs that are stabilized against nuclease degradation by chemical modifications are known to cause pro-inflammatory cytokine release.

It was surprising that the stimulation of latent retroviral infected cells did not result in an accelerated progression of the HIV infection to AIDS. It was not predictable that the stimulation with constructs according to the above disclosure enhances the success rates of HIC treatment with cART and/or HDACi. 

1.-21. (canceled)
 22. A method of treating a viral infection in a patient in need thereof, comprising administering to the patient a non-coding sequence of deoxyribonucleic acids comprising CCTAGGGGTT ACCACCTTCA TTGGAAAACG TTCTTCGGGG CGTTCTTAGG TGGTAACC CCTAGGGGTT ACCACCTTCA TTGGAAAACG TTCTTCGGGG CGTTCTTAGG TGGTAACC (SEQ ID NO:5).
 23. The method of claim 22, wherein the viral infection is a latent infection.
 24. The method of claim 22, wherein the viral infection is a retroviral infection.
 25. The method of claim 24, wherein the viral infection is a latent infection.
 26. The method of claim 22, wherein the viral infection is an HIV-1 infection.
 27. The method of claim 26, wherein the viral infection is a latent infection.
 28. The method of claim 22, wherein the non-coding sequence of deoxyribonucleic acids has a length of up to 200 nucleotides.
 29. The method of claim 22, wherein the non-coding sequence of deoxyribonucleic acids has a length of 116 nucleotides.
 30. The method of claim 22, wherein the non-coding sequence of deoxyribonucleic acids is administered in combination with combination antiretroviral therapy.
 31. The method of claim 30, wherein the non-coding sequence of deoxyribonucleic acids is administered simultaneously with combination antiretroviral therapy.
 32. The method of claim 30, wherein the non-coding sequence of deoxyribonucleic acids is administered alternating with combination antiretroviral therapy.
 33. The method of claim 30, wherein the non-coding sequence of deoxyribonucleic acids is administered successively with combination antiretroviral therapy. 